A simple light-activated proton pump occurs in the purple membrane, a differentiated crystalline membrane region of the extremely halophilic bacteria Halobacterium halobium. Bacteriorhodopsin, the only protein constituent of the membrane, vectorially pumps protons out of the cell, producing an electrochemical gradient that drives ATP synthesis. The objective of the proposed research is to understand the mechanism of action of the bacteriorhodopsin proton pump at the molecular level. The proposed experiments have three specific aims: 1) Complete characterization of steady-state light-induced proton release and uptake from isolated sheets of purple membrane (light-induced Bohr effect) as a function of: pH, ionic strength, ionic composition, detergent concentration, detergent composition, temperature, light intensity, wavelength, and protein concentration. The results, analyzed in terms of a simple 3-state model, will specify the conditions under which steady-state proton release or uptake may be used as an assay for proton pump activity. 2) Study of the effects of chemical modification on the proton pump, assayed by changes in steady-state proton binding. Covalent modifications will be directed against carboxyl, amino, acid side chains by proteolysis and isolation of modified peptides. Particular attention will be focused on a striking inhibitory effect of a carbodiimide recently discovered. This study will be unique in its use of steady-state light-induced proton release and uptake as an assay for pump activity. It will not be necessary to dissociate and reassemble the membranes, or prepare vesicles, or study secondary spectroscopic effects. The results will locate functionally important side chains in the three-dimensional structural model of bacteriorhodopsin. The nature of such groups and their location will suggest a molecular mechanism. Knowledge of the molecular mechanism of a proton pump will support efforts to understand oxidative phosphorylation, other ion pumps, and other active transport processes. The results will benefit ressarch on mitochondrial and bacterial metabolism, pharmacology and toxicology, and many areas of basic medical science.